Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both PKC and Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phosphoS473-Akt detected on western blots. Furthermore, the PKC specific inhibitor, GF109203X, increases IGF-1 induced phosphoS473-Akt and Akt kinase activity, and enhances IGF-1 protection from UVC induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduces phosphoS473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuates IGF-1 and epidermal growth factor (EGF) induced phosphoS473Akt, reduces Akt kinase activity, and blocks IGF-1 protection from UVC-induced apoptosis. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors. Overexpression and activation of PKC delta and epsilon reduced AktSer473 phosphorylation compared to control cells. In contrast, overexpression of PKC alpha enhanced phosphorylation of AktSer473. Our results suggest that activation of PKC delta and epsilon provides a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes, and serves as a modulator of cell survival pathways in response to external stimuli.Skin keratinocytes are major mediators of host immune responses. The skin is also a target for immunologically based inflammation in many pathological states. Activation of protein kinase c (PKC) can induce cutaneous inflammation, but the precise role of each of 6 cutaneous PKC isoforms (?, ?, ?, ?, ?, ?) that regulate normal skin homeostasis or contribute to skin pathology has not been clarified. We generated transgenic mice that overexpress PKC alpha in the basal layer of the epidermis and the outer root sheath of hair follicles under the regulation of the bovine keratin 5 promoter. K5-PKC alpha transgenic mice exhibit severe intraepidermal neutrophilic inflammation and disruption of the epidermis and upper hair follicles when treated topically with 12-0-tetradecanoylphorbol-13-acetate (TPA). Both TPA and UVB cause apoptosis in transgenic skin, but only TPA evokes intraepidermal inflammation. TPA also induces apoptosis in cultured transgenic keratinocytes, and this is prevented by an AP-1 dominant negative construct. However, inhibiting AP-1 in vivo does not abrogate intraepidermal inflammation. Transcripts for specific cytokines and chemokines are elevated in TPA treated cultured transgenic keratinocytes, and conditioned culture medium from these cells promotes neutrophil migration in vitro. Chemokine expression and neutrophil migration are not diminished by inhibiting AP-1. Thus, PKC alpha activation induces keratinocyte apoptosis via an AP-1 dependent pathway and mediates chemokine induction and intraepidermal inflammation independently. This model system will be useful to define specific chemokines regulated by PKC alpha that promote intraepidermal neutrophilic inflammation, a condition that characterizes several human cutaneous diseases such as pustular psoriasis and acute generalized exanthematous pustulosis.Activation of PKC alpha increases the production of TNF alpha and the transcription of chemotactic factors (MIP-2, KC, S100A8/A9), VEGF and GM-CSF in K5-PKC alpha keratinocytes. In response to PKC alpha activation, NF-kappaB translocates to the nucleus, and this is associated with IkappaB phosphorylation and degradation. Preventing IkappaB degradation reduces both the expression of inflammation associated genes and chemoattractant release. To determine if TNF alpha mediated NF-kappaB translocation and subsequent expression of pro-inflammatory factors, K5-PKC alpha mice were treated systemically with a dimeric soluble form of p75 TNF receptor (etanercept) or crossed with mice deficient for both TNF receptor (TNFR) isoforms, and keratinocytes were cultured in the presence of TNF alpha neutralizing antibodies. The in vivo treatment and TNFR deficiency did not prevent inflammation, and the in vitro treatment did not prevent NF-kappaB nuclear translocation after TPA. Together these results implicate PKC alpha as a regulator of a subset of cutaneous cytokines and chemokines responsible for intraepidermal inflammation independent of TNF alpha. PKC alpha inhibition may have therapeutic benefit in some human inflammatory skin disorders.To determine the function that protein kinase C alpha may play in skin tumor promotion, K5-PKC alpha mice were subjected to the two-stage skin tumor induction protocol. Initiation (7,12-dimethylbenzaanthracene, 100microg) and sub-optimal promotion with 12-O-tetradecanoyl-phorbol-13-acetate (TPA, 1microg/twice a week) leads to a 10 fold increase of both papilloma burden and tumor incidence in K5-PKC alpha mice at 15 weeks compared to the wildtype animals. Multiple TPA paintings in the absence of initiation cause a strong hyperplastic response in the transgenic epidermis. To test whether PKC alpha overexpression potentiates tumor growth independent of its inflammatory activity, primary K5-PKC alpha keratinocytes were initiated by transduction with the v-rasHa oncogene and grafted with normal fibroblasts onto a dorsal skin site of athymic recipient mice. Tumors evolving from initiated K5-PKC alpha keratinocytes grew to a larger size compared to their wildtype counterparts. The incipient tumor cell is maintained in a suppressed state in initiated skin, and this can be modeled in vitro by mixing an excess of normal keratinocytes with a small number of papilloma cells. Using this model, selective outgrowth of papilloma cells by TPA treatment is detected when K5-PKC alpha keratinocytes, but not wildtype keratinocytes, are the suppressing population. K5-PKC alpha keratinocytes express TNF alpha in response to TPA, and TNF alpha signaling is important in skin tumor promotion. However, bigenic K5-PKC alpha mice or bigenic keratinocytes doubly deficient for TNF alpha receptors (TNFR1-/-R2-/-) display the same TPA-mediated inflammatory response in vivo and the same apoptotic response in vitro as the single K5-PKCa mice and cells. Together these results suggest that the sensitivity of K5-PKC alpha transgenic skin to tumor formation is independent of the inflammatory response and TNF alpha induction but associated with pathways relating to reduced ability to suppress initiated keratinocytes and enhanced growth of neoplastic clones.Ingenol 3-angelate (Ing3A) is one of the active ingredients extracted from Euphorbia peplus. It has been used as a traditional medicine for treating warts, corns, skin cancer and other skin conditions. Ing3A has been shown to inhibit cell proliferation and induce apoptosis in a number of human tumor cell lines, including cells from colon and lung cancers. Recently, it was reported that topical application of Ing3A reduced the size of xenografted tumors in mice. Ing3A is structurally similar to tumor promoter phorbol ester and has been known to bind to and activate PKC in vitro. Like phorbol ester, Ing3A causes inflammation on the ears of mice and promotes tumor formation after DMBA initiation. Using two mouse skin tumor cell lines, 308 and PAM212, we studied the molecular targets of Ing3A and its effect on cellular function. Ing3A induces PKC phosphorylation, an indicator for activation. MAPK pathway proteins MEK1/2 and MAPK as well as PKC substrates MARCKS and Adducin are phosphorylated after Ing3A treatment.